DETERMINATION OF AFLATOXINS IN PEANUT (Arachis hypogaea L.) COLLECTED FROM KINSHASA, DEMOCRATIC REPUBLIC OF CONGO AND PRETORIA, SOUTH AFRICA: A COMPARATIVE STUDY by ILUNGA KAMIKA Submitted in accordance with the requirements for the degree of MASTER OF SCIENCE in the subject LIFE SCIENCES at the UNIVERSITY OF SOUTH AFRICA SUPERVISOR: Dr L S TEFFO Co-SUPERVISOR: Dr D R P KATERERE FEBRUARY 2012 1 Student number: 43544134 I hereby declare that the dissertation ‘Determination of aflatoxins in peanut (Arachis hypogaea L.) collected from Kinshasa, Democratic Republic of Congo and Pretoria, South Africa: a comparative study’ is my own work and that all the sources that I have used or quoted have been indicated and acknowledged by means of complete references. SIGNATURE DATE Ilunga Kamika © Unisa 2012 i ACKNOWLEDGEMENT With profound gratitude to the almighty, the most beneficent and merciful God, I would like to express my sincere appreciation to the following persons and organisations for their involvement in the successful completion of this project: My supervisors, Dr Snow Teffo and Dr David Katerere for their mentorship and kind support during my study. Thank you for the valuable input and for always steering me in the right direction. Without your guidance this study would not have been possible. Pamella Mngqawa, Dr Hester Vismer and Dr John Rheeder from PROMEC Unit, MRC, Tygerberg, Cape Town, for their technical support and guidance. Analysis of samples was carried out using HPLC and fluorometer from PROMEC Unit Laboratory, IKS (Health) Unit at Delft and Mpumalanga National Dept of Agriculture Laboratory. Thanks for allowing me to utilise your laboratory facilities for this project without which this project would not have been a success. SANPAD for funding this research and UNISA for the Postgraduate Bursary. Sizwe Ngubeni (Mpumalanga National Dept of Agriculture) for his technical assistance. Prof SR Moyo from Polytechnic of Namibia for the inputs he made during the initial stages of my study. My parents Mr and Mrs K. Kamika and all my siblings for their prayers, love and encouragement. Prof. M and Mrs M. Kalemba for their supports and prayers ii Finally, a special word of appreciation to those not cited above, for their love, inspiration, prayers and for believing in me. iii ABSTRACT This study assessed the mycological and aflatoxin contamination of peanuts collected from Kinshasa, DRC and Pretoria, South Africa. Forty peanut samples were collected randomly at informal markets in the two cities and analysed for mycoflora and aflatoxins (B1, B2, G1 and G2) using standard methods. The results indicated that 95% and 100% of peanut samples collected from Kinshasa and Pretoria, respectively were contaminated with aflatoxigenic fungi with Kinshasa’s samples being the most contaminated (up to 49, 000 CFU/g). Seventy percent (70 %) of Kinshasa-samples and 35% of Pretoria-samples exceeded the maximum allowable limit of aflatoxin B1 set by JECFA (5 ppb). Statistical evidence showed a significant positive correlation between mycoflora and aflatoxin level for Kinshasa- samples (r = 0.4743, p < 0.005) while Pretoria-samples showed no correlation. The study reveals that high level of contamination in Kinshasa-samples could be due to the tropical nature of the climate and poor storage conditions as compared to Pretoria which is sub-tropical and sanitary regulations are enforced. Key terms: Aspergillus; aflatoxigenic fungi; mycotoxin; aflatoxin; peanut; Kinshasa; Pretoria; DRC; South Africa; HPLC; fluorometer iv TABLE OF CONTENTS DECLARATION i ACKNOWLEDGEMENT ii ABSTRACT iv LIST OF FIGURES viii LIST OF TABLES x LIST OF ABBREVIATIONS xi CHAPTER ONE: INTRODUCTION 1 1.1. INTRODUCTION 1 1.2. PROBLEM STATEMENT 7 1.3. GENERAL AIM AND OBJECTIVES 8 1.4. SIGNIFICANCE OF THE STUDY 8 1.5. LIMITATION OF THE STUDY 9 1.6. CHAPTER LAYOUT 9 CHAPTER TWO: LITERATURE REVIEW 11 2.1. MYCOTOXINS 11 2.1.1. Aflatoxins 13 2.1.1.1. Chemistry of major aflatoxins 14 2.1.1.2. Aflatoxin-producing fungi 15 2.1.1.3. Biosynthesis of aflatoxin B1 18 2.1.1.4. Bioactivation of aflatoxin B1 19 2.1.2. Aflatoxin B1 and its health effects 22 2.1.2.1. Acute effects of aflatoxin B1 23 v 2.1.2.2. Chronic effects of aflatoxin B1 24 2.2. ECONOMIC IMPACT 34 2.3. NATURAL OCCURRENCE OF AFLATOXIN IN AFRICA 35 2.4. LEGISLATION 38 2.5. COMMON ANALYTICAL METHODS FOR AFLATOXIN ANALYSIS 41 CHAPTER THREE: MATERIALS AND METHODS 44 3.1. DESCRIPTION OF THE STUDY AREAS 44 3.1.1. Kinshasa 45 3.1.2. Pretoria 46 3.2. MYCOLOGICAL ANALYSIS 48 3.3. AFLATOXIN ANALYSIS IN PEANUT SAMPLES 49 3.3.1. Aflatoxin analysis using fluorometric method (VICAM) 49 3.3.1.1. Chemicals and reagents 49 3.3.1.2. Apparatus 50 3.3.1.3. Validation of VICAM analytical method 50 3.3.1.4. Sample preparation and aflatoxin determination using fluorometry 51 3.3.2. Aflatoxin analysis using HPLC system 52 3.3.2.1. Chemicals and reagents 52 3.3.2.2. Apparatus 52 3.3.2.3. Standard preparation 53 3.3.2.4. Validation of analytical method using the HPLC system 53 3.3.2.5. Determination of aflatoxin from peanut samples using HPLC 55 3.4. STATISTICAL ANALYSIS 56 vi CHAPTER FOUR: RESULTS AND DISCUSSIONS 57 4.1. MYCOLOGICAL ANALYSIS 57 4.2. AFLATOXIN ANALYSIS USING THE FLUOROMETRIC METHOD (VICAM) 59 4.2.1. Validation of the fluorometric method 59 4.2.1.1. Precision and recovery 59 4.2.1.2. Linearity validation 60 4.2.2. Determination of aflatoxin in raw peanut samples 60 4.3. AFLATOXIN ANALYSIS USING BY HIGH-PRESSURE LIQUID CHROMATOGRAPHY (HPLC) 62 4.3.1. Validation of analytical method using HPLC 62 4.3.1.1. Precision and recovery validation 62 4.3.1.2. Linearity validation 64 4.3.1.3. Specificity for HPLC method 65 4.3.2. Determination of aflatoxins in peanut samples using the HPLC system 66 CHAPTER FIVE: GENERAL DISCUSSION 74 CHAPTER SIX: CONCLUSIONS AND RECOMMENDATIONS 80 REFERENCES 82 vii LIST OF FIGURES Figure 2.1: Chemical structure of main aflatoxins 14 Figure 2.2: Scanning electron microscopy pictures of (a) A. parasiticus; and (b) A. flavus spores 16 Figure 2.3: Metabolism of AFB1 leading to reactive metabolites and biomarkers 20 Figure 2.4: Correlation between populations with high liver cancer rates and high risk of chronic aflatoxin exposure 25 Figure 2.5: Percentage of global population covered by mycotoxin regulation in 2003 40 Figure 2.6: African map highlighting the presence or absence of mycotoxin regulation (in most case aflatoxins) 40 Figure 3.1: Agro-ecological zones in Africa highlighting zones with high/low risk on fungal spoilage and aflatoxin contaminations 44 Figure 3.2: Sampling point situated in Ngaliema, Kinshasa, DRC 46 Figure 3.3: Sampling point situated at Marabastad, Pretoria, South Africa 48 Figure 4.1: An example of Pretoria ground peanut samples plated on AFPA and showing a growth of Aflatoxin-producing fungi 58 Figure 4.2: Standard curve highlighting the regression linear of fluorometric analysis 60 Figure 4.3: Liquid chromatograms of spiked peanut samples highlighting the recoveries of aflatoxin analogue at different concentrations 64 Figure 4.4: Standard curve highlighting the linear regression for the HPLC method 65 Figure 4.5: Liquid chromatograms highlighting the specificity of the HPLC method 66 viii Figure 4.6: Comparison of Aflatoxin and biomass level in peanut samples collected from Kinshasa and Pretoria 70 Figure 4.7: Comparison of aflatoxin level obtained using HPLC and VICAM method (fluorometric method) in peanut samples collected from Kinshasa and Pretoria 72 ix LIST OF TABLES Table 2.1: Environmental factors for Aspergillus growth and aflatoxin production 17 Table 2.2: A selection of tolerated levels of aflatoxin B1 in food 41 Table 3.1: Average temperature and precipitation of Kinshasa, DRC 45 Table 3.2: Average temperature and precipitation in Pretoria, South Africa 47 Table 4.1: Biomass level of aflatoxin-producing fungi in raw peanut samples collected from both Kinshasa and Pretoria 57 Table 4.2: Recoveries and precision of total aflatoxins spiked at different concentrations in clean peanut samples 59 Table 4.3: Average aflatoxin levels in peanut samples collected from Kinshasa and Pretoria 61 Table 4.4: Precision and recoveries of each aflatoxin analogue at different spiking levels 63 Table 4.5: Aflatoxin levels (B1, B2, G1 and G2) in raw peanut samples collected from Kinshasa and Pretoria 68 x LIST OF ABBREVIATIONS AFB1: aflatoxin B1 AFB2: aflatoxin B2 AFG1: aflatoxin G1 AFG2: aflatoxin G2 AFs: total aflatoxins ATA: alimentary toxic aleukia aw water activity unit DNA: deoxyribonucleic acid DRC: Democratic Republic of Congo DON: deoxynivalenol ELISA: enzyme-linked immunosorbent assay EU: European Union FAO: Food Agriculture Organization FB1: fumonisin B1 GDP: gross domestic product GNP: gross national product HBV: hepatitis B virus HBsAg: hepatitis B anti-gene HCC: hepatocellular carcinoma HCV: hepatitis C virus HIV/AIDS: human immunodeficiency virus/acquired immunodeficiency syndrome HPLC: high-pressure liquid chromatography xi IARC: International Agency for Research on Cancer IAC: immunoaffinity column IFAD: International Fund for Agricultural Development JECFA: Joint FAO/WHO Expert Committee on Food Additives RNA: ribonucleic acid TLC: thin-layer chromatography USA: United States of America WHO: World Health Organization ZEA: zearalenone xii CHAPTER ONE: INTRODUCTION 1.1. INTRODUCTION Developing countries, especially in Africa, face many socio-economic challenges of which poor food security and food safety are paramount (Schmidhuber and Tublello, 2007). The Food and Agriculture Organization (FAO) defines food security as a situation that exists when all people, at all times, have physical, social, and economic access to sufficient, safe, and nutritious food to meet their dietary needs and food preferences for an active and healthy life (Schmidhuber and Tublello, 2007).